Somatic gene therapy can be defined as the ability to program the expression of foreign genes in non-germ line (i.e., non-sperm and egg) cells of an animal. Methods of somatic gene therapy can be divided into two categories. Ex vivo gene therapy involving the removal of cells from a host organism, transfection a foreign gene into those cells, and reimplantation or transplantation of the transformed or transgenic cells back into a recipient host. In contrast, in vivo gene therapy involves transfection of a foreign gene directly into cells of a recipient host without the need for prior removal of those cells from the host.
The utility of somatic gene therapy for human subjects is dependent upon a number of factors. First, the transfection method must be efficient. Second, expression of the foreign gene should be localized to specific target tissues. Third, a given transfection process should be associated with a minimal risk of mutating the host cells and of causing a persistent infection of the host organism.
Several possible strategies to introduce genes into tissues of the body have been employed in the past (Stratford-Perricaudet et al., 1990; Rosenfeld et al., 1992; Wolfe et al., 1992). Procedures to introduce foreign genes into cells include direct transfection (Davis et al., 1986) and retroviral gene transfer (Dichek et al., 1991; Wilson et al., 1988a; Wilson et al., 1988b; Kay et al., 1992). In some cases, genetically altered cells have been reintroduced into animals (Dichek et al., 1991; Roy Chowdhury et al., 1991) where their continued function has been monitored for variable periods of time.
Recently, adenovirus-mediated gene transfer has been investigated as a means of somatic gene therapy into eukaryotic cells and into whole animals (van Doren et al., 1984a; van Doren et al., 1984b; Ghosh-Choudhury and Graham, 1987; Stratford-Perricaudet et al., 1990; Rosenfeld et al., 1991; Rosenfeld et al., 1992). A problem with adenovirus mediated gene transfer is the low level of gene product expression in target cells and a resultant lack of a functional effect.
Although adenovirus-mediated gene transfer has been used to treat ornithine transcarbamylase (OTC) deficiency in newborn mice, the expression of the ornithine transcarbamylase enzyme in the virus infected mice was typically at or below expression levels in normal mice with the result that the defect was only parally corrected (Stratford-Perricaudet et al., 1990). On the basis of those data, one would not expect that adenovirus-mediated gene transfer would be applicable to treatment of a disease requiring an overexpression of a gene product.
Adenovirus mediated transfer of the gene for cystic fibrosis transmembrane conductance regulator (CFTR) into the pulmonary epithelium of cotton rats has been attempted, although it has not been possible to assess the biological activity of the transferred gene because there was no physiologic effect of gene transfer despite expression of the CFTR protein in lung airway cells (Rosenfeld et al., 1992). Still further, lung expression of .alpha.1-antitrypsin protein was not associated with a physiologic effect (Rosenfeld et al., 1991). Taken together, those data do not demonstrate that adenovinus can transfer genes into cells and direct the expression of sufficient protein to achieve a physiologically relevant effect.
Targeting somatic gene therapy to cardiac tissue can be used in the treatment of a number of inherited and acquired cardiac diseases such as genetic disorders of myocardial cells. By way of example, injection of the normal dystrophin cDNA can be used to correct the defects in cardiac contractility seen in patients with Duchenne's muscular dystrophy. By way of further example, the injection of plasmids encoding recombinant angiogenesis factors directly into the left ventricular wall can be used to stimulate new collateral circulation in areas of chronically ischemic myocardium. Somatic gene therapy can also be used to directly study the molecular mechanisms regulating cardiac myocyte gene expression both during cardiac myogeneses and in a variety of pathophysiologic states such as cardiac hypertrophy.
As many as 1.5 million patients per year in the U.S. suffer a myocardial infarction (MI). Many millions more suffer from syndromes of chronic myocardial ischemia due to large and small vessel coronary atherosclerosis. Many of these patients will benefit from the ability to stimulate collateral vessel formation in areas of ischemic myocardium. Adenovirus mediator gene transfer methods provide an alternative approach to the current methods of coronary artery bypass and percutaneous transluminal coronary angioplasty. In particular, many patients have such revere and diffuse atherosclerosis that they are not candidates for CABG or PTCA. Thus far, there has been no approach which has successfully stimulated collateral vessel formation in areas of ischemic myocardium.
Previous approaches have all utilized in vitro transfection protocols into neonatal cardiocytes or transgenic approaches in mice. Such studies are complicated by the fact that neonatal cardiocytes may not reflect the in vivo situation and by the fact that neonatal cardiocytes have an extremely limited life span in tissue culture and cannot be incorporated into the heart. Moreover, transgenic approaches are lengthy (requiring 6 months to 1 year) technically difficult and expensive. The gene transfer approach of the present invention provides a solution to these problems and provides for the stable expression of recombinant gene products in cardiac myocytes in vivo.